Identification of specific tumor antigens recognized by cytotoxic T cells has greatly accelerated efforts to develop effective immunotherapies for cancer, but this knowledge has not yielded clinically effective strategies. The major impediments have been a subset of CD4+ regulatory T (Treg) cells that can negatively regulate effector T cells, thus diminishing their antitumor effects. Thus, the long-term goals of Project 4 are to develop and test in a clinical setting new strategies that will reverse the suppressive effects of Treg cells in patients with follicular lymphoma (FL). This broad objective will be pursued in four specific research aims suggested by the applicant's recent demonstration of high percentages of Treg cells in clinical samples of FL and a novel discovery that Toll-like receptors (TLRs) in Treg cells can be manipulated to reverse their suppressive function. Aim 1 seeks to identify and define discrete subsets of Treg cells in FL samples, and determine their mechanism(s) of immune suppression. Aim 2 will identify and characterize the natural ligands of antigen-specific FL-derived Treg cells to understand how such cells are activated and maintained at tumor sites. Aim 3 asks whether TLR signaling controls Treg cell suppressive function in follicular lymphoma as it does in melanoma and other tumors, and whether Poly-G nucleotides are indeed the optimal ligands for manipulating Treg cell function. A Phase I clinical trial is planned to demonstrate the feasibility of using TLR ligands as drugs to reduce or reverse Treg cell suppressive function in patients with FL. The major strengths of this application are (1) the availability of established CD4+ CD25+ Treg cell lines derived from different types of cancer including FL, (2) the development of all requisite technologies (some unique) needed to analyze Treg cell function and identify optimal TLR ligands, and (3) compelling preliminary data on the reversal of Treg cell function through TLR signaling, A positive outcome for this translational research project would open new opportunities for removing a major impediment to effective immunotherapy for follicular lymphoma and perhaps other types of tumors as well. Lay summary: Therapies designed to enhance the ability of a patient's immune system to attack cancer cells are often thwarted by a small subset of the patient's immune cells called Treg cells. This application aims to find ways to downregulate Treg cells so that the patient's immune system can effectively fight cancer.